Identification of a zinc-binding cystic fibrosis antigen in human saliva by 65Zn probing and N-terminal sequencing.
Human whole saliva was collected at 4 degrees C into a protein inhibitor cocktail to prevent proteolytic degradation. Saliva was clarified by centrifugation and fractionated by immobilized metal affinity chromatography (IMAC) after charging the column with zinc ions. Proteins with an affinity for zinc were eluted from the IMAC column, characterized by sodium dodecylsulphate-polyacrylamide gel electrophoresis, and probed with 65ZnCl2 after electrotransfer on to polyvinylidenefluoride membranes. A zinc-binding protein of approx. 11 kDa was characterized by N-terminal sequencing followed by a FASTA search of Genbank. The first 40 residues were sequenced, of which the first 34 residues were used to conduct a FASTA search and yielded and homology > 97% coding for sequences of mRNA of two proteins, a cystic fibrosis antigen with an M(r) of 10,938 and a calcium-binding inflammatory protein MRP8 with an M(r) of 10,835 expressed in chronic inflammation. The identity of the last 15 residues of the sequence and the likelihood that the protein is secreted via saliva indicates that the 11-kDa protein is the cystic fibrosis antigen, a protein not previously reported in saliva.
Davey
H P
HP
Department of Basic Dental Science, Dental School, University of Wales College of Medicine, Cardiff, U.K.
Embery
G
G
Creeth
J E
JE
Cummins
D
D
D000595:Amino Acid Sequence; D000943:Antigens, Differentiation / Q000302:isolation & purification / Q000378:metabolism; D000954:Antigens, Surface / Q000302:isolation & purification* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D040501:Calgranulin A; D002498:Centrifugation; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D039841:Leukocyte L1 Antigen Complex; D008297:Male; D008567:Membranes, Artificial; D008970:Molecular Weight; D019006:Neural Cell Adhesion Molecules / Q000302:isolation & purification* / Q000378:metabolism; D011145:Polyvinyls; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D019275:Radiopharmaceuticals; D012471:Salivary Proteins and Peptides / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate; D013501:Surface-Active Agents; D015032:Zinc / Q000378:metabolism*; D015037:Zinc Radioisotopes; D005465:Fluorocarbon Polymers
D000943:Antigens, Differentiation; D000954:Antigens, Surface; D002135:Calcium-Binding Proteins; D040501:Calgranulin A; D039841:Leukocyte L1 Antigen Complex; D008567:Membranes, Artificial; D019006:Neural Cell Adhesion Molecules; D011145:Polyvinyls; D012333:RNA, Messenger; D019275:Radiopharmaceuticals; D012471:Salivary Proteins and Peptides; D012967:Sodium Dodecyl Sulfate; D013501:Surface-Active Agents; D015032:Zinc; D015037:Zinc Radioisotopes; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers